Synthesis and antileishmanial activity of 1,2,4,5-tetraoxanes against leishmania donovani

Article abstract of DOI:10.3390/molecules25030465

A chemically diverse range of novel tetraoxanes was synthesized and evaluated in vitro against intramacrophage amastigote forms of Leishmania donovani. All 15 tested tetraoxanes displayed activity, with IC50 values ranging from 2 to 45 μm. The most active tetraoxane, compound LC140, exhibited an IC50 value of 2.52 ± 0.65 μm on L. donovani intramacrophage amastigotes, with a selectivity index of 13.5. This compound reduced the liver parasite burden of L. donovani-infected mice by 37% after an intraperitoneal treatment at 10 mg/kg/day for five consecutive days, whereas miltefosine, an antileishmanial drug in use, reduced it by 66%. These results provide a relevant basis for the development of further tetraoxanes as effective, safe, and cheap drugs against leishmaniasis.

Full text of DOI:10.3390/molecules25030465

molecules
Letter
Synthesis and Antileishmanial Activity of
1,2,4,5-Tetraoxanes against Leishmania donovani
Lília I. L. Cabral 1,2, Sébastien Pomel 3 , Sandrine Cojean 3, Patrícia S. M. Amado 1,2
,
Philippe M. Loiseau 3,* and Maria L. S. Cristiano 1,2,
*
1
Center of Marine Sciences, CCMAR, Gambelas Campus, University of Algarve, UAlg,
8005-139 Faro, Portugal; liliacabral80@gmail.com (L.I.L.C.); patricia.s.amado@gmail.com (P.S.M.A.)
Department of Chemistry and Pharmacy, Faculty of Sciences and Technology, FCT, Gambelas Campus,
University of Algarve, UAlg, 8005-139 Faro, Portugal
Chimiothérapie Antiparasitaire, Université Paris-Saclay, CNRS, BioCIS, 92290 Châtenay-Malabry, France;
sebastien.pomel@u-psud.fr (S.P.); sandrine.cojean@u-psud.fr (S.C.)
2
3
*
Correspondence: philippe.loiseau@u-psud.fr (P.M.L.); mcristi@ualg.pt (M.L.S.C.);
Tel.: +351-912-074-576 (M.L.S.C.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 23 December 2019; Accepted: 20 January 2020; Published: 22 January 2020
Abstract: A chemically diverse range of novel tetraoxanes was synthesized and evaluated in vitro
against intramacrophage amastigote forms of Leishmania donovani. All 15 tested tetraoxanes displayed
activity, with IC50 values ranging from 2 to 45
µm. The most active tetraoxane, compound LC140,
exhibited an IC50 value of 2.52 0.65 m on L. donovani intramacrophage amastigotes, with a selectivity
±
µ
index of 13.5. This compound reduced the liver parasite burden of L. donovani-infected mice by 37%
after an intraperitoneal treatment at 10 mg/kg/day for five consecutive days, whereas miltefosine,
an antileishmanial drug in use, reduced it by 66%. These results provide a relevant basis for the
development of further tetraoxanes as effective, safe, and cheap drugs against leishmaniasis.
Keywords: Leishmaniasis; antileishmanial chemotherapy; peroxide-derived antimalarials; tetraoxanes;
antimalarials re-purposing
1. Introduction
Leishmaniases are neglected diseases caused by protozoan parasites of the genus Leishmania and
transmitted by the bite of plebotomine sandflies. Visceral leishmaniasis, the most virulent among
leishmaniases, affects mostly tropical and subtropical areas of the world. However, it is spreading out of
these areas, namely along southern Europe [1,2]. As with other neglected and poverty-related diseases,
most patients suffering from leishmaniases do not benefit from a complete treatment, due to the high
cost of available drugs, the need for a long treatment period, low accessibility, an inadequate mode of
administration, and drug resistance [3]. These drawbacks have triggered a search for new treatment
methods, preferably based on recent technologies. The novel drugs for leishmaniasis should be potent
and effective, able to clear the parasite burden in a few days, active against resistant strains of Leishmania
donovani, orally available, safe, and affordable by the standards of the affected populations [4].
The increasing use of artemisinin and derivatives has clearly evidenced the potential of peroxides
in the treatment of vector-borne diseases [
Plasmodium sp. and have been used as antimalarials for around three decades, mostly in Artemisinin
Combination Chemotherapy (ACT) protocols [ ]. However, the high cost of artemisinin, associated
5]. Artemisinins were found to be active against all strains of
6
with the low yield of extraction from its natural source (Artemisia annua), together with some toxicity
and a short plasma half-life, leading to complex administration regimens or recrudescence, restricts
the therapeutic potential of artemisinins. In addition, recent findings of decreased clinical efficacy of
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ACTs in Southeast Asia due to resistance [
7] have raised concerns over the lifetime of this class as
antimalarials. In order to overcome these limitations while maintaining efficacy, various synthetic
analogues, incorporating the key peroxide pharmacophore of artemisinin, were developed [ ].
Among these, trioxolanes and tetraoxanes have shown activity against different parasites, such as
the protozoans Plasmodium spp. [10 19], Perkinsus spp. [20], and the parasitic flatworms Schistosoma
8,9
–
spp [21]. A main advantage of trioxolanes and tetraoxanes is their availability, due to straightforward
synthesis from inexpensive starting materials, enabling the preparation of chemically diverse libraries
of analogues and a better selection of a lead compound [22,23].
The use of artemisinin and its semi-synthetic derivatives for the treatment of leishmaniases
has been proposed by several authors [24–33]. Regarding the potential application of synthetic
endoperoxides with antimalarial properties, Cortes et al. [34] reported the antiparasitic activity
of a small selection of trioxolanes against promastigote and intramacrophage amastigote forms of
Leishmania infantum, at micromolar concentrations, introducing the relevance of synthetic endoperoxides
for antileishmanial chemotherapy. Given this observation of the antileishmanial activity of ozonide-type
antimalarials, it seemed logical to explore 1,2,4,5-tetraoxanes, which also incorporate the endoperoxide
core, although these compounds exhibit an enhanced thermodynamic stability compared with their
1,2,4-trioxolane [35,36] or 1,2,4-trioxane [37] counterparts. This singular thermodynamic stability
observed in 1,2,4,5-tetraoxanes was clarified by Gomes et al. [38] through theoretical calculations based
on stereoelectronic analysis, where the enhanced stability was attributed to a stereoelectronic ‘double
anomeric effect’ that stabilizes the six-membered ring system.
Therefore, our aim was to synthesize 1,2,4,5-tetraoxanes, analogues of the ozonides already
reported to have antileishmanial activity [31]. For comparison, we have also prepared novel
unsymmetrical 1,2,4,5-tetraoxanes and 1,2,4-trioxolanes with polar water-solubilizing groups (Table 1),
known to reduce neurotoxicity and increase the activity profiles, as reported in previous works based
on artemisinin derivatives [39]. In the present contribution, we disclose the low micromolar activity
of a range of peroxides comprising 15 tetraoxanes and two trioxolanes against intramacrophage
amastigote forms of L. donovani. The results are compared with those of dihydroartemisinin (DHA),
artesunate (ATS), and the antileishmanial drug miltefosine. From the tested tetraoxanes, compound
LC140 displayed a slight in vivo activity against L. donovani. It is worth noting that 1,2,4,5-tetraoxanes
are easily prepared, offering the possibility of new candidates with improved pharmacologic profiles.
Table 1. Inhibitory concentrations (IC50) of artemisinin derivatives, synthetic 1,2,4-trioxolanes,
1,2,4,5-tetraoxanes, and miltefosine (control) against intramacrophage amastigote forms of Leishmania
donovani LV9, evaluation of cytotoxicity (CC50) against the macrophage cell line RAW 264.7, selectivity
index (SI), and estimated ClogP values.
Activity
IC50 ± SD
(µm)
Toxicity
CC50 ± SD
(µm)
Entry Compounds
Structures
SI
ClogP value a
(A)
DHA
3.07 ± 0.45
>75.00
>24.00
2.59
(B)
(C)
ATS
15.00 ± 0.63
16.30 ± 2.41
>75.00
>75.00
5.00
2.68
4.66
LC129
>4.00

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Table 1. Cont.
Activity
Toxicity
CC50 ± SD
(µm)
Entry Compounds
Structures
IC50 ± SD
SI
ClogP value a
(µm)
(D)
LC136
18.36 ± 4.97
55.20 ± 6.30
3.00
3.14
(E)
(F)
(G)
LC137
LC139
LC140
7.75 ± 1.12
13.17 ± 0.03
2.52 ± 0.65
43.15 ± 3.25
>50.00
5.50
>3.80
13.50
4.52
2.91
3.19
34.12 ± 5.38
(H)
(I)
LC141
LC146
LC153
18.36 ± 3.19
16.00 ± 1.05
17.33 ± 2.02
>100.00
>100.00
>50.00
5.40
5.24
3.59
3.64
>6.20
>2.80
(J)
(K)
(L)
LC159
LC163
44.49 ± 1.13
12.16 ± 3.96
ND
ND
4.39
3.41
>50.00
>4.10
(M)
(N)
(O)
(P)
LC165
LC167
LC177
LC179
LC180
8.79 ± 1.79
14.97 ± 0.07
17.57 ± 0.85
29.05 ± 0.26
28.97 ± 1.95
>75.00
>50.00
>50.00
ND
>8.00
>3.30
>2.80
ND
3.35
4.67
4.89
4.16
3.76
(Q)
ND
ND
(R)
(S)
(T)
PA5
PA6
19.22 ± 0.11
17.74 ± 2.78
0.71 ± 0.20
>50.00
>50.00
>2.60
>2.80
76.40
4.83
4.53
4.22
HePC
(Miltefosine)
54.30 ± 2.14
a
ND—not determined. Calculated using ALOGPS software (http://www.vcclab.org/lab/alogps/).
2. Results and Discussion
All tested peroxides showed antiproliferative activity against intramacrophage amastigote forms
of L. donovani, exhibiting IC50 values in a range from 2 to 45 m and clearly demonstrating the
susceptibility of L. donovani parasites to the peroxide chemotype (Table 1, entries A–T). Overall,
the values are higher than those obtained for miltefosine (0.71 0.20 m, Table 1, entry T), using the
µ
±
µ
same parasite strain and similar experimental conditions, but the tetraoxanes appeared generally to
be less toxic. Among the tested tetraoxanes, three compounds exhibited an IC50 value lower than
10
µ
m (compounds LC140, LC137, and LC165; 2.52
±
0.65, 7.75
±
1.12, and 8.79
±
1.79 µm, respectively;
Table 1, entries G, E, and M), LC165 being significantly less toxic than miltefosine. Interestingly,

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compounds LC137 and LC140 may be obtained from commercially available materials with only two
synthetic steps.
Our results indicate that the activities shown by tetraoxanes and trioxolanes with a close chemical
structure are similar. For the two tested trioxolanes, LC129 and LC136, the IC50 values ranged between
16.30
±
2.41 and 18.36
±
4.97 µm (Table 1; entries C, D). These values are similar to those obtained
for tetraoxanes LC163, LC177, and PA5 (Table 1; entries L, O, and R). The IC50 value obtained for
tetraoxane LC165 is also very close to that previously reported for the corresponding trioxolane [34].
From our results, it is possible to conclude that the chemical nature of the cyclohexyl substituent
has an impact on activity, for both tetraoxanes and trioxolanes. However, due to the relatively narrow
range of values, differences are possibly related to variations in ClogP, with different drug uptakes
and pharmacokinetics. Quite interestingly, the activities of all of the artemisinin-derived compounds
assayed also lie in the low micromolar range. DHA and tetraoxane LC140 exhibited similar IC50 values
(3.07
±
0.45
µ
m and 2.52
±
0.65 µm), respectively (Table 1; entry A and Table 1; entry G), while the
more polar ATS was shown to be slightly less active (15.00 ± 0.63 µm (Table 1; entry B)).
Our results showed that the activities exhibited by the synthetic tetraoxanes LC137, LC140, and
LC165 are similar to those of the semisynthetic artemisinin derivatives (DHA and ATS), disclosing the
potential of tetraoxanes to be anti-proliferative agents against intramacrophage amastigote forms of
L. donovani. The peroxide bridge in the synthetic compounds should play a role in the mechanism of
action, as seen for artemisinin and its semisynthetic derivatives. It has been observed that artemisinin
mediates its toxicity against Leishmania promastigotes by inducing a redox imbalance following
the generation of reactive oxygen species (ROS) secondary to cleavage of its endoperoxide bridge,
the process terminating in a caspase-independent, apoptotic mode of cell death [25,28,29,33]. It is
important to highlight that 1,2,4,5-tetraoxanes have been reported to possess a higher stability and
better antimalarial activity compared to their ozonide counterparts [37]. In this work, we can observe
that both classes of endoperoxides exhibit similar anti-leishmanial activities, though better IC50 values
in tetraoxanes LC137, LC140, and LC165 were observed. Future studies for comparison of metabolic
properties should be considered.
Concerning the in vivo antileishmanial evaluation, the treatment regimen at a dose of 10 mg/kg/day,
for five consecutive days, corresponds to the classical flowchart used by Drugs for Neglected Diseases
Initiative (DNDi), the non-governmental organization (NGO) in charge of drug development against
Neglected Diseases. DNDi considers further development for a compound only if a significant activity
can be demonstrated under these stringent conditions.
In vivo, we observed that one mouse among eight mice died in each treated batch (LC137 and
LC140) one day after the last treatment. This datum is in relation to a toxicity, whereas no other apparent
signs of toxicity were observed (Figure 1). At this early stage, no deeper investigation was performed at
the toxicological level. Under these conditions, only the in vivo activity of miltefosine was statistically
significant, with a reduction of 66% of the parasite burden in the liver, whereas compound LC140
reduced the liver parasite burden by 37% (Table 2). These results justify further pharmacomodulations
in order to optimize this series and to obtain a better in vivo effect at 10 mg/kg/day for five consecutive
days, these regimen conditions being sine qua non to go further.
Table 2. In vivo activity of tetraoxanes LC137 and LC140, and miltefosine, a reference antileishmanial
drug, on L. donovani/Balb/C mice. Compounds were administered intraperitonially, at a dose of 10
mg/kg/day, for five consecutive days.
% reduction of
Parasite Burden in
the Liver
Number of
Mice
Number of
Dead Mice
LDU (108) ±
Batch
Treatment Regimen
SD
LC137 10 mg/kg
LC140 10 mg/kg
Miltefosine
8
8
8
1
1
0
0
10 mg/kg/d x5 (IP)
10 mg/kg/d x5 (IP)
10 mg/kg/d x5 (IV)
3.92 ± 1.03
2.95 ± 0.69
1.60 ± 0.45
4.70 ± 0.71
16.40
37.29
65.9
-
Control
10
Treated with the excipient

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Figure 1. In vivoactivityoftetraoxanesLC137andLC140, andmiltefosine, areferenceantileishmanialdrug.
3. Materials and Methods
The structures of the 15 tetraoxanes assayed against intramacrophage amastigote forms of
L. donovani differ only in the chemical nature of the cyclohexyl substituent (Table 1). From this library,
only compounds L137 [40], LC140 [14], L153 [41], and LC163 [19] were previously reported in the
context of antiparasitic chemotherapy (compound LC163 was disclosed by our group). For comparative
purposes, we have also evaluated the activity of a small library of 1,2,4-trioxolanes and that of the
known peroxide-based antiplasmodial drugs dihydroartemisinin (DHA) and artesunate (ATS) (Table 1).
The 1,2,4,5-tetraoxanes and 1,2,4-trioxolanes were synthesized by adapting procedures described in
the literature. The synthetic procedures and experimental details for the preparation and chemical
characterization of compounds are included in the Supplementary Materials. The inhibitory effect
of each compound tested against L. donovani is expressed as IC50 (concentration of drug inhibiting
parasite growth by 50%), according to a protocol previously described [42]. The cytotoxicity was
evaluated on the mouse monocyte/macrophage cell line RAW 264.7, as the parasite host cells, and is
expressed as CC50 (cytotoxic concentration inhibiting the cell growth by 50%) following a previously
described protocol [42]. The selectivity index (SI) is defined as the ratio CC50/IC50. Miltefosine was
used as reference drug. Results are compiled in Table 1.
For in vivo evaluation, all procedures involving animals were conducted in compliance with
the standards for animal experiments and were approved by the local committee for animal care
(0858.01/2014, Versailles, France). The protocol of evaluation on the L. donovani/Balb/C mice model is
presented by Morais et al. [43] Two 1,2,4,5-tetraoxane derivatives were evaluated by an intraperitoneal
route at 10 mg/kg/day on five consecutive days. Miltefosine, as the control, was evaluated at the same
dose by an intravenous route. Animals were sacrificed three days after the end of treatment. Livers
and spleens were weighed and drug activity was estimated microscopically by counting the number of
amastigotes/500 liver cells in Giemsa-stained impression smears to calculate the Leishmania donovani
units (LDUs) for liver parasite burdens, using Stauber’s formula. The mean number of parasites per
gram of liver among treatment groups and controls was compared. Three independent counts were
performed and the results are expressed as the mean values
groups and controls were compared using the Kruskal-Wallis nonparametric analysis of variance test
±
SD. The parasite burden of treatment
for comparing two groups. Significance was established for a p value <0.05.

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4. Conclusions
The results presented herein unveil the potential of tetraoxanes as anti-proliferative agents against
intramacrophage amastigote forms of L. donovani. Compounds LC137, LC140, and LC165 (Table 1)
appear to be the most promising, combining a comparatively high activity and low toxicity. In vivo
,
LC140 appears to be a lead to investigate further through new pharmacomodulations (see Table 2).
Our data indicate that tetraoxanes and trioxolanes with a close chemical structure exhibit similar
activity. Also, the nature of the substituents attached to the endoperoxide core (tetraoxane or trioxolane)
appears to have a relatively modest effect on activity. Major aspects (accumulation, bioactivation, targets
involved, etc.) underlining the action of the tested compounds (including artemisinin derivatives)
require a deep and pluridisciplinary investigation, to unravel the mode of action of these compounds.
Supplementary Materials: The following are available online, S.1: Synthetic procedures and experimental details for
the synthesis and chemical characterization of compounds. S.1.1: General methods and analytical techniques. S.1.2:
Preparation of intermediate building blocks. S.1.2.1: Preparation of 3-chloro-1,2-benzisothiazole-1,1-dioxide. S.1.2.2:
Preparation of 1-phenyl-1H-tetrazol-5(4H)-one, 1-methyl-1H-tetrazole-5-amine and 2-methyl-2H-tetrazole-5-amine.
S.1.2.3: Preparation of tert-butyl(4-aminobutyl)carbamate, LC64. S.1.3: Synthetic route to trixolanes. S.1.3.1:
Synthesis of 1,2,4-trioxolanes LC129 and LC136. S.1.4: Synthetic route to tetraoxanes. S.1.4.1: Synthesis of
1,2,4,5-tetraoxanes. S.2: Spectra of the compounds. S3. In vitro antileishmanial screening. S3.1: Cell lines and
cultures. S3.2: In vitro antileishmanial evaluation on intramacrophage amastigotes. S3.3: In vitro antileishmanial
evaluation on Leishmania donovani axenic amastigotes. S3.4: Evaluation of compounds cytotoxicity. S4. In vivo
antileishmanial screening. S4.1: Animal and housing. S4.2: Evaluation of in vivo acute toxicity by an intraperitoneal
route. S4.3: In vivo antileishmanial evaluation. S.5: References.
Author Contributions: Conceptualization: L.I.L.C., P.S.M.A., S.C., and M.L.S.C.; methodology (synthesis of the
compounds): L.I.L.C. and P.S.M.A.; methodology (in vitro tests): S.C.; (in vivo tests): S.P.; validation: M.L.S.C. and
P.M.L.; formal analysis: M.L.S.C. and P.M.L.; investigation: L.I.L.C., P.S.M.A., S.C., M.L.S.C., and P.M.L.; resources:
M.L.S.C. and P.M.L.; writing—original draft preparation: L.I.L.C., P.S.M.A., S.C., and M.L.S.C.; writing—review
and editing: M.L.S.C. and P.M.L.; project administration: M.L.S.C.; funding acquisition: M.L.S.C. and P.M.L.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Fundaç
ão para a Ciência e a Tecnologia (FCT), and FEDER/COMPETE 2020-UE,
through projects UID/Multi/04326/2019 (Centre of Marine Sciences-CCMAR) and PTDC/MAR-BIO/4132/2014.
Acknowledgments: The authors gratefully acknowledge Fundaç o para a Ci ncia e a Tecnologia (FCT), and
ã
ê
FEDER/COMPETE 2020-UE, through projects UID/Multi/04326/2019 (Centre of Marine Sciences-CCMAR) and
PTDC/MAR-BIO/4132/2014. L lia I. L. Cabral and Patr cia S. M. Amado thank CCMAR and FCT for fellowships
í
í
(from project PTDC/MAR-BIO/4132/2014 and grant SFRH/BD/130407/2017, respectively).
Conflicts of Interest: The authors declare no conflicts of interest.
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